| Polygonum multiflorum Thunb. (Chinese name He-shou-wu, HSW) is originated from the root of Polygonum multiflorum, a member of Polygonaceae, and traditionally used as Chinese herbal medicine in raw state or processed form. Radix Polygoni Multiflori (RPM), and Radix Polygoni Multiflori Praeparata (RPMP), and HSW-containing products, as Shou-Wu-Pian or Shou-Wu-Wan, are widely used around the world. However, hepatic adverse effect, even poisoning death cases, associated with HSW products have been increasing since 1990s. The problem of HSW hepatotoxicity caused high attention of both at home and abroad. Upon to date, the underlying hepatotoxicity mechanisms and contens of HSW are incompletely understood, seriously restricted the clinical application of HSW and threaten the patient’s medication safety.In order to better understand the hepatotoxicity of HSW, a 3D in vitro toxicity testing model was developed by magnetic levitation method for culture of the human hepatoma cell line HepG2, and acetaminophen (APAP), a typical hepatotoxic drug, was specially used as the positive control. HepG2 cells cultured in this model re-acquire the ability of storage of glycogen, and show increased expression of drug metabolism enzymes, drug transporters, nuclear receptors and liver-specific marker compared to 2D monolayer cultures. Although 3D HepG2 cells can make adaptive response to these drugs under low concentrations, but 3D HepG2 cells only show higher sensitivity with APAP when compared with 2D HepG2 cells, which is more obvious with 7d repeated doses, while 2D HepG2 cells are more sensitive to RPM and RPMP, suggest that the hepatotoxicity of HSW may be related to the body’s metabolism ability.The major mechanisms of hepatotoxicity include inhibition of mitochondrial function, disruption of REDOX balance, glutathione (GSH) depletion, activation of stress response pathways, such as apoptosis, oxidative stress response, endoplasmic reticulum stress response, et al. Multi-parametric assays, particularly imaging-based analysis approaches like high-content screening (HCS) technology, may be the best predictive tool for application of the above mechanistic understanding the hepatotoxicity of HSW. The preliminary study results show that both RPM and RPMP have certain liver toxicity, and RPM shows more severe toxicity than RPMP; RPM and RPMP can both cause a certain amount of oxidative stress and endoplasmic reticulum stress; and reduce the mitochondrial membrane potential, damaging the mitochondrial function; RPM causes oxidative stress response in the earlier time, and endoplasmic reticulum stress later, while RPMP is completely opposite. Application UPLC/TOF-MS technology to study the material foundation of RPM and RPMP, we found that after processing of RPM, the content of 2,3,5,4’-tetrahydroxystilbene-2-O-(3-D-glucoside (TSG) and emodin-8-O-β-D-glucoside (emodin-G) fell by 37% and 78% respectively, and the content of emodin rose by about 337%, indicating the hepatotoxicity mechanism of HSW is directly linked to is chemical composition.With the 3D magnetic levitation HepG2 spheroids culture, we studied the toxicity of HSW in both 2D and 3D cells model the first time. In addition, we compared the differences of material foundation between RPM and RPMP with UPLC/TOF-MS technology, and assessed the hepatotoxicity of RPM and RPMP by measuring a panel of signals directly linked to key mechanisms of liver injury using HCS technology for the first time. |
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